Vaccine Formulations Comprising Saponin-containing Adjuvants

ABSTRACT

The present invention provides for a novel oil-in-water (O/W) emulsion, with increased stability in the presence of bacterial or viral suspensions, especially those concentrated and non-purified (crude extracts) or minimally purified. The emulsion of the present invention can act as vehicle for the delivery of a pharmaceutical composition comprising at least one immunogen and, in particular, an immunogen selected from the group consisting of an inactivated pathogen, an attenuated pathogen, a subunit, a recombinant expression vector, and a plasmid or combinations thereof.

INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional patent applicationNo. 61/241,171, filed on Sep. 10, 2009, and further makes reference tothe following patent applications: U.S. patent application Ser. No.12/027,776, filed on Feb. 7, 2008, U.S. patent application Ser. No.10/899,181, filed on Jul. 26, 2004, now granted as U.S. Pat. No.7,371,395, and U.S. provisional patent application No. 60/490,345, filedon Jul. 24, 2003. The foregoing applications, and all documents citedtherein or during their prosecution (“applicant cited documents”) andall documents cited or referenced in the applicant cited documents, andall documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention.

FIELD OF THE INVENTION

The present invention relates to oil-in-water emulsions, their use asadjuvants, and pharmaceutical, immunologic, or vaccine compositionscomprising the same.

BACKGROUND

The use of adjuvants in vaccines is well known. An adjuvant is acompound that, when combined with a vaccine antigen, increases theimmune response to the vaccine antigen as compared to the responseinduced by the vaccine antigen alone. Among strategies that promoteantigen immunogenicity are those that render vaccine antigensparticulate, those that polymerize or emulsify vaccine antigens, methodsof encapsulating vaccine antigens, ways of increasing host innatecytokine responses, and methods that target vaccine antigens to antigenpresenting cells (Nossal, 1999, In: Fundamental Immunology. Paul (Ed.),Lippincott-Raven Publishers, Philadelphia, Pa.; Vogel and Powell, 1995,In: Vaccine Design. The Subunit and Adjuvant Approach. Powell and Newman(Eds.), Plenum Press, NY, N.Y. p. 141). Because of the essential roleadjuvants play in improving the immunogenicity of vaccine antigens, theuse of adjuvants in the formulation of vaccines has been virtuallyubiquitous (Nossal, 1999, supra; Vogel and Powell, 1995, supra; see alsoPCT publication WO 97/18837, the teachings of which are incorporatedherein by reference). Conventional adjuvants, well-known in the art, arediverse in nature. They may, for example, consist of water-insolubleinorganic salts, liposomes, micelles or emulsions, i.e. Freund'sadjuvant. Other adjuvants may be found in Vogel and Powell, 1995,mentioned supra. Although there is no single mechanism of adjuvantaction, an essential characteristic is their ability to significantlyincrease the immune response to a vaccine antigen as compared to theresponse induced by the vaccine antigen alone (Nossal, 1999, supra;Vogel and Powell, 1995, supra). In this regard, some adjuvants are moreeffective at augmenting humoral immune responses; other adjuvants aremore effective at increasing cell-mediated immune responses (Vogel andPowell, 1995, supra); and yet another group of adjuvants increase bothhumoral and cell-mediated immune responses against vaccine antigens(Vogel and Powell, 1995, supra).

Generally, emulsions used in vaccine formulation comprise a mixture ofoil, aqueous solution and surfactants. Some emulsions incorporate alipophilic surfactant such as SPAN 80® and a hydrophilic surfactant suchas TWEEN 80®.

However, problems of stability can be observed with emulsions used asvaccine adjuvants, in particular during storage or transport. This isparticularly true when these compositions contain concentratedimmunogens, especially non-purified concentrated immunogens. Typically,this is the case with adjuvants used in inactivated (killed) vaccines.This problem is even more significant with multivalent vaccinecompositions because the immunogens are more concentrated in the samevolume of diluent.

Another problem with adjuvant use is linked to a risk of adverse eventssuch as toxicity or local inflammation at the site of injection. Forexample, a local inflammatory response and/or granulomae may resultafter injection. In order to limit such an adverse reaction, surfactantsand other components in the emulsion may be reduced; however, thereduction may then result in a decrease in the stability of the vaccinecomposition. There is, therefore, a need for novel adjuvants and vaccinecompositions containing such adjuvants with increased safety andstability.

SUMMARY OF THE INVENTION

In a first embodiment the present invention provides for a noveloil-in-water (O/W) emulsion, with increased stability in the presence ofbacterial or viral suspensions, especially those concentrated andnon-purified or weakly purified.

Another embodiment of the present invention provides for a stable, safeand easily administrable, in particular injectable, O/W emulsion actingas a vehicle for the delivery of a pharmaceutical composition comprisingat least one active ingredient that may be, more particularly, animmunogen.

Yet another embodiment of the present invention provides for a stable,safe and injectable O/W emulsion acting as an adjuvant to increase theimmune response induced by an immunogen. In particular, the presentinvention provides a novel adjuvant which, when used in a vaccinecomposition containing an immunogen increases the vaccinate's cellularimmune response, humoral immune response or, preferably, both to theimmunogen.

Yet another embodiment of the present invention provides a stable, safeand immunogenic composition or vaccine comprising an O/W emulsion.

A further embodiment of the present invention provides for a method ofmaking a vaccine composition using the adjuvant of the instantinvention; the vaccine composition so obtained; and methods of usingthereof.

Still another embodiment of the present invention provides for a kitcomprising one or more vials. In one embodiment, the kit comprises onevial containing the adjuvant of the present invention and an immunogenor other pharmaceutical product. In yet another embodiment, the kitcomprises an immunogen or other pharmaceutical product in a first vial,and an adjuvant made according to the present invention in a secondvial, with the adjuvant designed to be mixed with the immunogen or othervaccine product before use.

In one embodiment, the present invention provides for an injectableoil-in-water (O/W) emulsion comprising:

-   -   (1) an aqueous solution comprising an immunogen;    -   (2) an aqueous solution comprising a hydrophilic ionic        surfactant such as saponin    -   (3) an optional aqueous solution comprising aluminum hydroxide    -   (4) a mineral oil;    -   (5) a non-ionic lipophilic surfactant;    -   (6) a non-ionic hydrophilic surfactant having a low HLB value        which comprises ethoxylated fatty acid diesters of sorbitan        (generally having HLB value between 11 and 13).

In another embodiment, the present invention provides for an injectableoil-in-water (O/W) emulsion comprising:

-   -   (1) an aqueous solution comprising an immunogen;    -   (2) an aqueous solution comprising a hydrophilic ionic        surfactant such as saponin    -   (3) a mineral oil;    -   (4) a non-ionic lipophilic surfactant;    -   (5) a non-ionic hydrophilic surfactant having a low HLB value        which comprises ethoxylated fatty acid diesters of sorbitan        (generally having HLB value between 11 and 13).

In another embodiment, the present invention provides for an injectableoil-in-water (O/W) emulsion comprising:

-   -   (1) an aqueous solution comprising an immunogen;    -   (2) an aqueous solution comprising a hydrophilic ionic        surfactant such as saponin    -   (3) an optional aqueous solution comprising aluminum hydroxide    -   (4) a non-ionic hydrophilic surfactant having a high        hydrophilic-lipophilic balance (HLB) value greater than 13 and        less than 40, in particular HLB≧13.5, and preferably HLB≧14;    -   (5) a mineral oil;    -   (6) a non-ionic lipophilic surfactant;    -   (7) a non-ionic hydrophilic surfactant having a low HLB value        (HLB value of about 9 to about 13).

In another embodiment, the present invention provides for an injectableoil-in-water (O/W) emulsion comprising:

-   -   (1) an aqueous solution comprising an immunogen;    -   (2) an aqueous solution comprising a hydrophilic ionic        surfactant such as saponin    -   (3) a non-ionic hydrophilic surfactant having a high        hydrophilic-lipophilic balance (HLB) value greater than 13 and        less than 40, in particular HLB≧13.5, and preferably HLB≧14;    -   (4) a mineral oil;    -   (5) a non-ionic lipophilic surfactant;    -   (6) a non-ionic hydrophilic surfactant having a low HLB value        (HLB value of about 9 to about 13).

In yet another embodiment, the present invention provides for a vaccinecomposition comprising a novel emulsion containing at least oneimmunogen suitable for eliciting an immunologic response in a vaccinate.The invention further provides such compositions wherein the emulsionacts as an adjuvant to increase the immune response induced by theimmunogen, in particular, to increase the cellular response, the humoralresponse or preferably both.

In another embodiment the present invention provides for a method ofmaking a vaccine composition wherein an immunogen, especially animmunogen in dry form, which can be obtained, for example, bylyophilization or by vitrification, or in an aqueous solution,especially wherein said dry form or said aqueous solution additionallycomprises an ionic surfactant, for example saponin, and optionallyadditionally comprises aluminum hydroxide, is mixed with the adjuvantaccording to the instant invention. The immunogen may be selected fromthe group consisting of: inactivated pathogens, attenuated pathogens,sub-unit antigens, purified antigens, unpurified antigens, or antigensproduced recombinantly using bacterial, yeast, plant, insect, or animalcells, expression vectors including plasmids, and the like. The antigensmay be purified by means well-known in the art including, but notlimited to, ultrafiltration, ultracentrifugation, size-exclusiongel-filtration, ion-exchange chromatography, and PEG-purification. Thepathogen may be bacterial, viral, protozoal, or fungal in origin or theimmunogen may constitute an antitoxin.

In another embodiment, the present invention provides for a method ofinducing an immune response in a vaccinate against a pathogen comprisingadministering the vaccine composition of the present invention to thevaccinate.

In another embodiment, the present invention provides for kitscomprising a single vial containing purified immunogens and the emulsionaccording to the instant invention. In one such embodiment, theimmunogens contained within the single vial comprise purified FMD virusantigens.

In another embodiment, the present invention provides for kitscomprising at least two vials, in a first vial an immunogen, especiallyan immunogen in dry form or in solution in an aqueous medium, especiallywherein said dry form or said aqueous solution additionally comprises anionic surfactant, advantageously saponin, and optionally additionallycomprises aluminum hydroxide, and in a second vial an adjuvant oremulsion according to the present invention. The use of kits thatcomprise at least two vials is particularly effective in cases where thecombination of the discrete components (i.e. the mixture into a singlevial of the contents of the at least two vials) would result in avaccine formulation of reduced stability.

It is noted that in this disclosure and particularly in the claims,terms such as “comprises”, “comprised”, “comprising” and the like canhave the meaning attributed to such terms in U.S. Patent law; e.g., theycan mean “includes”, “included”, “including”, and the like; and thatterms such as “consisting essentially of and “consists essentially ofhave the meaning ascribed to them by U.S. Patent law, e.g., they allowfor elements not explicitly recited, but exclude elements that are foundin the prior art or that affect a basic or novel characteristic of theinvention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof, to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification, includingreference to the accompanying figures, wherein:

FIG. 1 provides graphs of the Phase Inversion Temperature (PIT)determination (measured by conductivity) for the vaccine formulations oftrials 1 & 2 on days 7, 22, 121, and 330 (one year stability study). ThePIT determination is one measure of vaccine formulation stability.

FIG. 2 provides graphs of the PIT determination for the vaccineformulations of trials 3 & 4 on multiple days after production (one yearstability study);

FIG. 3 provides graphs of the PIT determination for the vaccineformulations of trials 5 & 6 on multiple days after production (one yearstability study);

FIG. 4 provides graphs of the PIT determination for the vaccineformulations of trials 7 & 8 on multiple days after production (one yearstability study);

FIG. 5 provides a graph of the PIT determination for the vaccineformulations of trial 9 on multiple days after production (one yearstability study);

FIG. 6 provides graphs of the PIT determination for the vaccineformulations (of trials 1-9) produced according to the instantinvention, and stored for 36 months;

FIG. 7 provides a graph that indicates the time-dependent changes in therectal temperature of pigs treated according to the materials andmethods disclosed in Example 6;

FIG. 8 provides a graph that indicates the maximum temperature changeobserved for pigs treated according to the materials and methodsdisclosed in Example 6;

FIG. 9 provides a graph that indicates vaccine potency (PD50) versuspayload (μg) for pigs treated according to the materials and methodsdisclosed in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

Other objects, features and aspects of the present invention aredisclosed in, or are obvious from, the following Detailed Description.It is to be understood by one of ordinary skill in the art that thepresent discussion is a description of exemplary embodiments only and isnot intended as limiting the broader aspects of the present invention,which broader aspects are embodied in the exemplary construction. Infact, it will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Forinstance, features illustrated or described as part of one embodimentcan be used in another embodiment to yield a still further embodiment.It is intended that the present invention cover such modifications andvariations as come within the scope of the appended claims and theirequivalents. The contents of all references, published patents, andpatents cited throughout the present application are hereby incorporatedby reference in their entirety.

For convenience, certain terms employed in the Specification, Examples,and appended Claims are collected here.

As used herein, the term “animal” includes all vertebrate animalsincluding humans. It also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages. In particular, theterm “vertebrate animal” includes, but not limited to, humans, canines(e.g., dogs), felines (e.g., cats); equines (e.g., horses), bovines(e.g., cow, cattle), porcine (e.g., pigs), as well as in avians. As usedherein, the term “cow” or “cattle” is used generally to refer to ananimal of bovine origin of any age. Interchangeable terms include“bovine”, “calf', “steer”, “bull”, “heifer”, “cow” and the like.Interchangeable terms include “piglet”, “sow” and the like. The term“avian” as used herein refers to any species or subspecies of thetaxonomic class ava, such as, but not limited to, chickens (breeders,broilers and layers), turkeys, ducks, a goose, a quail, pheasants,parrots, finches, hawks, crows and ratites including ostrich, emu andcassowary. The term “pig” or “piglet” means an animal of porcine origin,while “sow” refers to a female of reproductive age and capability.

As used herein, the term “virulent” means an isolate that retains itsability to be infectious in an animal host.

As used herein, the term “inactivated vaccine” means a vaccinecomposition containing an infectious organism or pathogen that is nolonger capable of replication or growth. The pathogen may be bacterial,viral, protozoal or fungal in origin. Inactivation may be accomplishedby a variety of methods including freeze-thawing, chemical treatment(for example, treatment with formalin), sonication, radiation, heat orany other convention means sufficient to prevent replication or growthof the organism while maintaining its immunogenicity.

As used herein, the term “immunogenicity” means capable of producing animmune response in a host animal against an antigen or antigens. Thisimmune response forms the basis of the protective immunity elicited by avaccine against a specific infectious organism.

As used herein, the term “immune response” refers to a response elicitedin an animal. An immune response may refer to cellular immunity (CMI);humoral immunity or may involve both. The present invention alsocontemplates a response limited to a part of the immune system. Forexample, a vaccine composition of the present invention may specificallyinduce an increased gamma interferon response.

As used herein, the term “antigen” or “immunogen” means a substance thatinduces a specific immune response in a host animal. The antigen maycomprise a whole organism, killed, attenuated or live; a subunit orportion of an organism; a recombinant vector containing an insert withimmunogenic properties; a piece or fragment of DNA capable of inducingan immune response upon presentation to a host animal; a protein, apolypeptide, a peptide, an epitope, a hapten, or any combinationthereof. Alternately, the immunogen or antigen may comprise a toxin orantitoxin.

As used herein, the term “multivalent” means a vaccine containing morethan one antigen whether from the same species (i.e., different isolatesof FMD virus serotypes), from a different species (i.e., isolates fromboth Pasteurella hemolytica and Pasteurella multocida), or a vaccinecontaining a combination of antigens from different genera (for example,a vaccine comprising antigens from Pasteurella multocida, Salmonella,Escherichia coli, Haemophilus somnus and Clostridium).

As used herein, the term “adjuvant” means a substance added to a vaccineto increase a vaccine's immunogenicity. The mechanism of how an adjuvantoperates is not entirely known. Some adjuvants are believed to enhancethe immune response by slowly releasing the antigen, while otheradjuvants are strongly immunogenic in their own right and are believedto function synergistically. Known vaccine adjuvants include, but arenot limited to, oil and water emulsions (for example, complete Freund'sadjuvant and incomplete Freund's adjuvant), Corynebacterium parvum,Bacillus Calmette Guerin, aluminum hydroxide, glucan, dextran sulfate,iron oxide, sodium alginate, Bacto-Adjuvant, certain synthetic polymerssuch as poly amino acids and co-polymers of amino acids, saponin,“REGRESSIN” (Vetrepharm, Athens, Ga.), “AVRIDINE”(N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine), paraffinoil, muramyl dipeptide and the like.

As used herein, the term “emulsion” refers to a combination of at leasttwo substances, wherein a first substance is dispersed in a secondsubstance in which the first substance is insoluble. One example of anemulsion of the present invention is an oil phase dispersed in anaqueous phase.

As used herein, the term “incomplete emulsion” refers to a compositionto which at least one additional component must be added to make the“complete emulsion”. As used herein, the term “complete emulsion” can beconsidered equivalent to the “ready-to-use” immunological composition ofthe present invention. An example of a complete emulsion is animmunological composition according to the present invention that isready to be administered to an animal according to the methods of thepresent invention.

As used herein, the terms “pharmaceutically acceptable carrier” and“pharmaceutically acceptable vehicle” are interchangeable and refer to afluid vehicle for containing vaccine antigens that can be injected intoa host without adverse effects. Suitable pharmaceutically acceptablecarriers known in the art include, but are not limited to, sterilewater, saline, glucose, dextrose, or buffered solutions. Carriers mayinclude auxiliary agents including, but not limited to, diluents,stabilizers (i.e., sugars and amino acids), preservatives, wettingagents, emulsifying agents, pH buffering agents, viscosity enhancingadditives, colors and the like.

As used herein, the term “vaccine composition” includes at least oneantigen or immunogen in a pharmaceutically acceptable vehicle useful forinducing an immune response in a host. Vaccine compositions can beadministered in dosages and by techniques well known to those skilled inthe medical or veterinary arts, taking into consideration such factorsas the age, sex, weight, species and condition of the recipient animal,and the route of administration. The route of administration can bepercutaneous, via mucosal administration (e.g., oral, nasal, anal,vaginal) or via a parenteral route (intradermal, intramuscular,subcutaneous, intravenous, or intraperitoneal). Vaccine compositions canbe administered alone, or can be co-administered or sequentiallyadministered with other treatments or therapies. Forms of administrationmay include suspensions, syrups or elixirs, and preparations forparenteral, subcutaneous, intradermal, intramuscular or intravenousadministration (e.g., injectable administration) such as sterilesuspensions or emulsions. Vaccine compositions may be administered as aspray or mixed in food and/or water or delivered in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, or the like. The compositions can containauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, adjuvants, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standardpharmaceutical texts, such as “Remington's Pharmaceutical Sciences,”1990 may be consulted to prepare suitable preparations, without undueexperimentation.

The term “purified” as used herein does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedimmunogen preparation, such as protein or inactivated virus, is one inwhich the immunogen is more enriched than the immunogen is in itsnatural environment. An immunogen preparation is herein broadly referredto as “purified” such that the immunogen represents at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 98%, ofthe total immunogen content of the preparation. A “crude preparation”,which represents the lowest degree of purification, may contain aslittle as less than 60%, lest than 20%, less than 10%, less than 5%, orless than 1% of immunogenic components.

The term “highly purified” as used herein is intended to suggest a“higher degree of purity” as compared to the term “moderately purified”.This “higher degree of purity” can include, but is in no way limited to,reduced percentages of contaminants, in an immunological preparationthat has been “highly purified” versus an immunological preparation thathas been “moderately purified”. As discussed herein, “highly purified”immunological preparations will have the lowest to undetectablepercentages of contaminants that can cause: reduced desired immuneresponse, increased undesired immune response (e.g. hypersensitivityreaction), or reduced formulation stability. Similarly, an immunologicalpreparation that has been “moderately purified” contains relativelyreduced percentages of contaminants versus an immunological preparationthat has been “minimally purified”, which likewise, has reducedpercentages of contaminants versus a preparation designated a “crudepreparation”.

Contaminants in an immunological preparation can include, but are in noway limited to, substances that contribute negatively to animmunological composition according to the present invention. One ofseveral examples of a contaminant contributing negatively would be acontaminant that reduces the ability of an immunological composition ofthe present invention to elicit an immune response in animals.

Varying levels of purity (e.g. “highly purified”, “moderately purified”,and the like) can be achieved using various methods. For example, acombination of chromatography and size exclusion gel filtration canresult in a “highly purified” or “moderately purified” immunologicalpreparations. Differences in source/type of immunogens, as well asslight variations in purification procedures can significantly affectthe final degree of immunogen purity. In general, as used herein,immunological preparations having the lowest to highest percentage ofcontaminants will be described as 1) “highly purified, 2) “moderatelypurified”, 3) “minimally purified”, 4) “crude preparation”,respectively. A “highly purified” preparation will have the lowest levelacross all types of contaminants. A “moderately purified” preparationwill have relatively low levels of most types of contaminants, but mayhave one type of contaminant in higher abundance than would be observedfor a comparable “highly purified” preparation. Likewise, a “minimallypurified preparation” will have relatively low levels of some types ofcontaminants, but may have more than one type of contaminant in higherabundance than a comparable “moderately purified” preparation. Asexpected, a “crude preparation” has the highest level of contaminants,across all contaminant types, as compared to the other types ofpreparations discussed herein.

The present invention provides a novel oil-in-water (O/W) emulsioncomprising:

-   -   (1) an aqueous solution comprising a vaccine antigen or        immunogen capable of inducing an immune response in a host;    -   (2) an aqueous solution comprising an ionic surfactant;    -   (3) a non-ionic hydrophilic surfactant;    -   (4) a mineral oil;

In one embodiment, the novel oil-in-water (O/W) emulsion comprises:

-   -   (1) an aqueous solution comprising a vaccine antigen or        immunogen capable of inducing an immune response in a host;    -   (2) an aqueous solution comprising an ionic surfactant such as        saponin;    -   (3) a non-ionic hydrophilic surfactant having a        hydrophilic-lipophilic balance (HLB) value of greater than 13        and less than 40 (HLB>13, in particular HLB≧13.5, and preferably        HLB≧14);    -   (4) a mineral oil;    -   (5) a non-ionic lipophilic surfactant; and    -   (6) a non-ionic hydrophilic surfactant having a low HLB value        (HLB value between 9 and 13).

In another embodiment the present invention provides a noveloil-in-water (O/W) emulsion comprising:

-   -   (1) an aqueous solution comprising a vaccine antigen or        immunogen capable of inducing an immune response in a host;    -   (2) an aqueous solution comprising an ionic surfactant such as        saponin    -   (3) an optional aqueous solution comprising aluminum hydroxide    -   (4) a non-ionic hydrophilic surfactant having a        hydrophilic-lipophilic balance (HLB) value of greater than 13        and less than 40 (HLB>13, in particular HLB≧13.5, and preferably        HLB≧14);    -   (5) a mineral oil;    -   (6) a non-ionic lipophilic surfactant; and    -   (7) a non-ionic hydrophilic surfactant having a low HLB value        (HLB value between 9 and 13).

Some emulsions made according to the present invention are based on acombination of at least four (4) surfactants chosen among the members offour different groups of surfactants, and it is possible to use one ormore surfactant pertaining to each group. Three (3) of these groupscomprise non-ionic surfactants and one (1) of these groups comprisesionic surfactants, for example saponins.

In one of several embodiments, the concentration of the ionic surfactant(2) in the emulsion (in the present specification this means the finalemulsion comprising all ingredients unless otherwise indicated) is fromabout 0.01% to about 10%.

In one of several embodiments, the concentration of non-ionichydrophilic surfactant (7) in the emulsion (in the present specificationthis means the final emulsion comprising all ingredients unlessotherwise indicated) is from 1% to 8%, in particular from 1.5% to 6%,preferably from 2% to 5%, more preferably from 2.5% to 4%, expressed asa percentage in weight by volume of emulsion (w/v).

This group of surfactants comprises non-ionic hydrophilic surfactantshaving a low HLB value (HLB value between 9 and 13). This group includesbut is not limited to ethoxylated fatty acid monoester of sorbitan (inparticular 5 ethoxyl groups) (e.g. ethoxylated sorbitan monooleate suchas TWEEN 81®, ethoxylated fatty acid diesters of sorbitan, ethoxylatedfatty acid triesters of sorbitan (in particular 20 ethoxyl groups) (e.g.ethoxylated sorbitan trioleate such as TWEEN 85®), ethoxylated sorbitantristearate such as TWEEN 65®, ethoxylated fatty alcohols (in particular5-10 ethoxyl groups) (e.g. BRIJ 76®, BRIJ 56®, BRIJ 96®), ethoxylatedfatty acids (in particular 5-10 ethoxyl groups) (e.g. Simulsol 2599®,MYRJ 45®), ethoxylated castor oil (in particular 25-35 ethoxyl groups)(e.g. ARLATONE 650®, ARLATONE G®), and combinations thereof.

Ethoxylated fatty acid diesters of sorbitan and ethoxylated fatty acidtriesters of sorbitan are preferred, as well combinations of bothspecies. The fatty acid is preferably selected from the group consistingof oleate, palmitate, stearate, isostearate, laurate and thecombinations thereof. Preferred ethoxylated fatty acid triester ofsorbitan comprise ethoxylated sorbitan trioleate such as TWEEN 85®), orethoxylated sorbitan tristearate such as TWEEN 65®.

In one of several embodiments, the concentration of non-ionichydrophilic surfactant (4) is generally from 0.1% to 1.5%, in particularfrom 0.2% to 1.4%, preferably from 0.3% to 1.3%, more preferably from0.4% to 1.2%, expressed as a percentage in weight by volume of emulsion(w/v).

This second group of surfactants comprises non-ionic hydrophilicsurfactants having a high hydrophilic-lipophilic balance (HLB) value(HLB>13, in particular HLB≧13.5, and preferably HLB≧14). This groupcomprises ethoxylated fatty acid monoesters of sorbitan (in particular20 ethoxyl groups) (e.g. ethoxylated sorbitan monolaurate such as TWEEN20®, ethoxylated sorbitan monopalmitate such as TWEEN 40®, ethoxylatedsorbitan monostearate (such as TWEEN 60®, ethoxylated sorbitanmonooleate such as TWEEN 80®, ethoxylated fatty alcohols (in particular15-30 ethoxyl groups) (e.g. BRIJ 78®, BRIJ 98®, BRIJ 721®), ethoxylatedfatty acids (in particular 15-30 ethoxyl groups) (e.g. MYRJ 49®, MYRJ51®, MYRJ 52®, MYRJ 53®), non-ionic block-copolymers (e.g.polyoxyethylene/polyoxypropylene copolymer (POE-POP), such as LUTROLF127®, LUTROL F68®), and combinations thereof.

For the non-ionic block-copolymers, the percentages may be lower and bein particular from 0.1% to 0.5%, more particularly from 0.2% to 0.4%(weight by volume of emulsion (w/v)).

Preferred surfactants (4) comprise ethoxylated fatty acid monoesters ofsorbitan, such as those described above.

In one of several embodiments, the concentration of non-ionic lipophilicsurfactant (6) is from 0.1% to 2.5%, in particular from 0.2% to 2%,preferably from 0.2% to 1.5%, more preferably from 0.2% to 1.2%,expressed as a percentage in weight by volume of emulsion (w/v).

This group of surfactants comprises fatty acid esters of sorbitan (e.g.sorbitan monolaurate, like SPAN 20®, sorbitan monopalmitate, such asSPAN 40®, sorbitan monostearate, such as SPAN 60®, sorbitan tristearate,such as SPAN 65®, sorbitan monooleate, like SPAN 80®, sorbitantrioleate, like SPAN 85®, sorbitan monoisostearate, such as ARLACEL987®, sorbitan isostearate, such as CRILL 6®), fatty acid esters ofmannide (e.g. MONTANIDE 80®, mannide monooleate (such as ARLACEL A®),mannide dioleate, mannide trioleate, mannide tetraoleate), ethoxylatedfatty acid esters of mannide (2, 3 or 4 ethoxyl groups) (e.g. MONTANIDE888®, MONTANIDE 103®, ethoxylated mannide monooleate, ethoxylatedmannide dioleate, ethoxylated mannide trioleate, ethoxylated mannidetetraoleate), and combinations thereof.

The fatty acid is preferably selected from the group consisting ofoleate, palmitate, stearate, isostearate, laurate and combinationsthereof.

Preferred surfactants (6) comprise the fatty acid esters of sorbitan, inparticular those described above, and combinations thereof.

The surfactants of the invention may have fatty acids from animal orvegetal origin. The change of one origin for the other (for exampleanimal TWEEN 80® to vegetal TWEEN 80®) could be done simply with onlyminor adjustment in the formulation of the emulsion.

An emulsion according to the invention may have an overall concentrationof surfactants, by weight per volume of emulsion, from 1.2% to 10%, inparticular from 2% to 8%, preferably from 3% to 7%, more preferably from4% to 6%.

Generally, the emulsion according to the invention may have a phaseinversion temperature (PIT) which is ≧33° C., in particular ranges from33° C. to 65° C., more particularly from 36° C. to 60° C., preferablyfrom 37° C. to 55° C., and more preferably from 38° C. to 50° C.

The PIT is the temperature at which a water-in-oil emulsion changes toan oil-in-water emulsion or de-phases (breaks of the emulsion andseparation of the 2 phases). The PIT value may be measured by variousmeans, like for example by visual appearance (e.g. see example 2) or byconductivity. The emulsion is placed at a temperature below the PIT ofthe emulsion, for example of about 25° C. in a water-bath. Thetemperature is progressively increased. The change of the visual aspectof the emulsion is observed in comparison with a control emulsion,notably the fluidity, the viscosity, the separation in two phases, thechange of the surface aspect due to the migration of the oily phase tothe surface. The temperature, for which this change of visual aspect wasobserved, is the PIT value of the emulsion. Alternatively, the PIT isdetermined by the quick passage from a conductivity value of about 5-8milliSiemens/centimetre (mS/cm) (oil-in-water emulsion) to a value ofabout 0 mS/cm (water-in-oil emulsion) measured by a probe placed intothe emulsion, near its surface. The temperature, for which thetransition was observed, is the PIT value of the emulsion. One ofordinary skill n the art will be able to determine combinations ofsurfactants and oil, including their respective concentrations, in orderto produce emulsions according to the invention, and in particularemulsions having a PIT value within the ranges defined above withoutundue experimentation.

Generally, emulsions according to the present invention may contain, byvolume per volume of emulsion, from 3% to 55% of oil, in particular from5% to 50% of oil, preferably from 10% to 40% of oil and, morepreferably, from 20% to 40% of oil. By definition, ranges of values inthe present specification include always the limit of the range, unlessotherwise indicated.

The oil used may be a mineral oil including, but not limited to,paraffin oil such as isoparaffinic oil and/or naphtenic oil, squalane,pristane, polyisobutene oil, hydrogenated polyisobutene oil, polydeceneoil, polyisoprene oil, polyisopropene oil and the like. One advantageousmineral oil useful in the present invention may include an oilcomprising a linear or ramified carbon chain having a number of carbonatoms greater than 15, preferably from 15 to 32, and free of aromaticcompounds. Such oils may, for example, be those marketed under the name“MARCOL 52®” or “MARCOL 82®” (produced by Esso, France) or “DRAKEOL6VR®” or “DRAKEOL 5®” “DRAKEOL 7®” (produced by Penreco, USA),“CLEAROL®” (produced by Sonneborn, USA), “Paraffin Oil Codex AAB2®”(produced by Aiglon, France), BLANDOL (produced by Sonneborn, USA),ONDINA 915 (produced by Shell, UK).

The oil may also be a mixture of oils comprising at least 2 oilsselected among the oils described herein, and in any proportion. Themixture of oils may also comprise at least one oil selected among theoils described above and at least one vegetable oil, and this vegetableoil represents from about 0.1% to about 33% of the oily phase,preferably from about 10% to about 25% v/v. These vegetable oils areunsaturated oils rich in oleic acid that are biodegradable andpreferably liquid at the storage temperature (about +4° C.) or at leastmake it possible to give emulsions that are liquid at this temperature.For example the vegetable oil may be groundnut oil, nut oil, sunfloweroil, safflower oil, soya oil, onager oil and the like.

In one of several embodiments, hydrophilic surfactants (4) and (7)preferably include surfactants having the same hydrophilic part of themolecules. For instance, use is made of ethoxylated fatty acid esters ofsorbitan for each of hydrophilic surfactants (4) and (7). For example ifTWEEN 85® is chosen as non-ionic hydrophilic surfactants having a lowHLB value, the non-ionic hydrophilic surfactant having a high HLB valuewill advantageously have a hydrophilic part constituted with anethoxylated sorbitan, such as TWEEN 80®.

Generally, the present invention envisions using an aqueous solutioncomprising a suitable veterinary or pharmaceutically acceptable vehicle,excipient, or diluent including, but not limited to, sterile water,physiological saline, glucose, buffer and the like. The vehicle,excipient or diluent may also include polyols, glucids or pH bufferingagents. The vehicle, excipient or diluent may, for example, alsocomprise amino acids, peptides, antioxidants, bactericide, andbacteriostatic compounds. The aqueous solution is added to the oil andthe surfactants in quantity to obtain 100% of the volume of the emulsionaccording to the invention.

The hydrophilic-lipophilic balance (HLB) of an emulsion allows for theestimation of the hydrophilic or lipophilic force of a surfactant. TheHLB of an amphiphilic molecule is generally calculated as follow:

${HLB} = \frac{\left( {20 \times {weight}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {hydrophilic}\mspace{14mu} {part}} \right)}{\left( {{weight}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {amphiphilic}\mspace{14mu} {molecule}} \right)}$

The HLB may have a value ranging from 0 (for the most lipophilicmolecule) to 20 (for the most hydrophilic molecule). According to thechemical composition of the surfactant (notably for example the additionof ethoxyl groups or of alkene oxides), this estimation may change andthe domain of HLB value may increase (for example, the LUTROL F68® has aHLB of 29). With a mixture of surfactants, the HLB of the mixture is theaddition of the HLB of each surfactant, balanced by its weight ratio:

${HLB} = \frac{\begin{pmatrix}{{HLB}\mspace{14mu} {surfactant}\mspace{14mu} X \times} \\{{weight}\mspace{14mu} {surfactant}\mspace{14mu} X}\end{pmatrix} + \begin{pmatrix}{{HLB}\mspace{14mu} {surfactant}\mspace{14mu} Y \times} \\{{weight}\mspace{14mu} {surfactant}\mspace{14mu} Y}\end{pmatrix}}{\left( {{{weight}\mspace{14mu} {surfactant}\mspace{14mu} X} + {{weight}\mspace{14mu} {surfactant}\mspace{14mu} Y}} \right)}$

In one embodiment of an emulsion made according to the presentinvention, the final HLB of the emulsion is from about 9 to about 12,preferably from about 9.5 to about 11.5 and more preferably from about10 to about 11.5.

The present invention contemplates an emulsion comprising a paraffin oil(in particular at a concentration of from about 10% to about 40% andpreferably from about 20% to about 40%, expressed as a volume per volumeof emulsion (v/v)); a sorbitan fatty acid monoester (as non-ioniclipophilic surfactant), an ethoxylated fatty acid triester of sorbitan(as non-ionic hydrophilic surfactant having a low HLB value); and anethoxylated fatty acid monoester of sorbitan (as non-ionic hydrophilicsurfactant having a high HLB value). In particular the sorbitan fattyacid monoester is a sorbitan monooleate (in particular at theconcentration from 0.2% to 1.5%, preferably from 0.2% to 1.2% expressedas a weight per volume of emulsion (w/v)), the ethoxylated fatty acidtriester of sorbitan is an ethoxylated trioleate of sorbitan (inparticular at the concentration from 2% to 5%, preferably from 2.5% to4% w/v)) and the ethoxylated fatty acid monoester of sorbitan is anethoxylated sorbitan monooleate (in particular at the concentration from0.3% to 1.3%, preferably from 0.4% to 1.2% w/v). For example theemulsion comprises the paraffin oil at about 29.3% by volume per volumeof emulsion, the sorbitan monooleate at 0.6% by weight per volume ofemulsion, the ethoxylated trioleate of sorbitan at 3.4% by weight pervolume of emulsion, and the ethoxylated sorbitan monooleate at 0.75% byweight per volume of emulsion.

In a second embodiment according to the present invention, the emulsioncomprises a paraffin oil (in particular at a concentration from 10% to40%, preferably from 20% to 40% v/v), a sorbitan fatty acid monoester(as non-ionic lipophilic surfactant), an ethoxylated fatty acid triesterof sorbitan (as non-ionic hydrophilic surfactant having a low HLBvalue), and a non-ionic block-copolymer (as non-ionic hydrophilicsurfactant having a high HLB value). In particular the sorbitan fattyacid monoester is a sorbitan monooleate (in particular at theconcentration from 0.2% to 1.5%, preferably from 0.2% to 1.2% w/v), theethoxylated fatty acid triester of sorbitan is an ethoxylated trioleateof sorbitan (in particular at the concentration from 2% to 5%,preferably from 2.5% to 4% w/v) and the non-ionic block-copolymer is apolyoxyethylene/polyoxypropylene polymer (POE-POP) (in particular at theconcentration from 0.1% to 0.5%, preferably from 0.2% to 0.4% w/v). Forexample the emulsion comprises the paraffin oil at about 29.3% v/v, thesorbitan monooleate at 0.6% w/v, the ethoxylated trioleate of sorbitanat 3.4% w/v, and the ethoxylated sorbitan monooleate at 0.25% w/v.

In a particular embodiment, the invention contemplates an injectableoil-in-water (O/W) emulsion comprising:

-   -   (1) an aqueous solution comprising an active ingredient such as        a drug or an immunogen, preferably an immunogen;    -   (2) an aqueous solution comprising saponin    -   (3) a mineral oil;    -   (4) a non-ionic lipophilic surfactant; and    -   (5) a non-ionic hydrophilic surfactant having a low HLB value        which comprises of an ethoxylated fatty acid diester of sorbitan        (which may have a HLB value between 11 and 13).

In another particular embodiment, the invention contemplates aninjectable oil-in-water (O/W) emulsion comprising:

-   -   (1) an aqueous solution comprising an active ingredient such as        a drug or an immunogen, preferably an immunogen;    -   (2) an aqueous solution comprising saponin    -   (3) an aqueous solution comprising aluminum hydroxide    -   (4) a mineral oil;    -   (5) a non-ionic lipophilic surfactant; and    -   (6) a non-ionic hydrophilic surfactant having a low HLB value        which comprises of an ethoxylated fatty acid diester of sorbitan        (which may have a HLB value between 11 and 13).

An emulsion according to this embodiment comprises ethoxylated fattyacid diesters of sorbitan that may contain up to 20 ethoxy groups. Thefatty acids may be from animal or vegetable origin and may be selectedfrom the group consisting of oleate, palmitate, stearate, isostearate,laurate and combinations thereof. In one embodiment the ethoxylatedfatty acid is preferably oleate. The other ingredients, as well as thegeneral properties of the emulsion such as the PIT, may have the samecharacteristics than those described above.

Preferably, surfactant (6) comprises ethoxylated fatty acid diesters ofsorbitan, such as ethoxylated sorbitan dioleate, ethoxylated sorbitandistearate or ethoxylated sorbitan diisostearate, ethoxylated sorbitandipalmitate, ethoxylated sorbitan dilaurate, and combinations thereof.

Optionally other compounds may be added as co-adjuvants to the emulsion,including, but not limited to, alum; CpG oligonucleotides (ODN), inparticular ODN 2006, 2007, 2059, or 2135 (Pontarollo R. A. et al., Vet.Immunol. Immunopath, 2002, 84: 43-59; Wernette C. M. et al., Vet.Immunol. Immunopath, 2002, 84: 223-236; Mutwiri G. et al., Vet. Immunol.Immunopath, 2003, 91: 89-103); polyA-polyU (“Vaccine Design The Subunitand Adjuvant Approach”, edited by Michael F. Powell and Mark J. Newman,Pharmaceutical Biotechnology, 6: 03); dimethyldioctadecylammoniumbromide (DDA) (“Vaccine Design: The Subunit and Adjuvant Approach”,edited by Michael F. Powell and Mark J. Newman, PharmaceuticalBiotechnology, volume 6: 157),N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)propanediamine (such asAVRIDINE®) (Ibid, p. 148), carbomer, chitosan (see U.S. Pat. No.5,980,912 for example).

The present invention also provides a method of making a vaccinecomposition or immunologic composition comprising at least one antigenor immunogen composition and an adjuvant or emulsion made according tothe present invention. The antigen or immunogen composition may beincorporated during emulsion formation or, in an alternate embodiment,the antigen or immunogen composition, preferably additionally comprisingsaponin and optionally additionally comprising aluminum hydroxide, maybe added to the emulsion later as, for example, just before use.

The entire amount of the aqueous solution used may be present in theemulsion first produced. Or it may be that only a part of this aqueoussolution is used to form the emulsion, and the remaining quantity ofaqueous solution is added after incorporation of the immunogen. Theimmunogen or antigen may be in a dry form or present in some otherappropriate solid form and then mixed with the emulsion or, alternately,the antigen may be in solution, in particular in an aqueous solution,and this solution mixed with the emulsion.

Surfactants are preferably added to either the oil or the aqueoussolution according to their solubility. For example, the non-ioniclipophilic surfactants are added to the oil according to the inventionwhile non-ionic hydrophilic surfactants having a high HLB value areadded to the aqueous solution.

The emulsification can be prepared according to conventional methodsknown to one of ordinary skill in the art. For example, in oneembodiment of the present invention, the emulsion can be prepared at atemperature below the PIT of the emulsion, in particular at roomtemperature, e.g. at about 25° C. The aqueous phase and the oily phaseare mixed together by a mechanical agitation, e.g. with a turbineequipped with a rotor-stator able to create a high shearing force.Preferably the agitation starts at a low rotation speed and slowlyincreases in relation to the progressive addition generally of theaqueous solution in the oil. Preferably the aqueous solution isprogressively added to the oil. The ratio of oil/aqueous solution may beadapted to obtain a water-in oil (W/O) emulsion, for example, at aconcentration of about 40% to about 55% of oil (v/v). When the agitationis stopped, the emulsion changes progressively to an O/W emulsion (phaseinversion). After inversion and if needed, the emulsion is diluted byaddition of an aqueous solution to obtain the desired concentration ofoil into the final emulsion. The emulsion may be stored at about 5° C.

In another embodiment, the emulsion can be prepared at a temperaturehigher than the PIT of the emulsion. In a first step, the aqueous phaseand the oily phase are mixed together at a temperature higher than thePIT of the emulsion. Preferably the aqueous solution is progressivelyadded to the oil. The ratio of oil/aqueous solution may be adapted toobtain a water-in oil (W/O) emulsion, for example at a concentration ofabout 40% to about 55% of oil (v/v). The emulsification may be done byan agitation with low or no shearing force, e.g. with a static mixer ora marine helix or with a turbine at a very low rotation speed. Theemulsion obtained is a water-in-oil (W/O) emulsion. In a second step,the emulsion is cooled progressively below the PIT. During this step,the emulsion changes to an O/W emulsion (phase inversion). Afterinversion and if needed, the emulsion is diluted by addition of anaqueous solution to obtain the desired concentration of oil into thefinal emulsion. The emulsion may be stored at about 5° C.

The size of the droplets in the emulsion may be from about 100 nm toabout 500 nm. The emulsion may be used, for example, as an adjuvant toformulate a vaccine composition or a pharmaceutical composition. Theemulsion may also be used as a solvent to dissolve a dried product,especially a dry product containing, for example, attenuatedmicroorganisms or live recombinant vectors.

In a particular embodiment, a pre-emulsion is produced with only a partof the aqueous solution. This pre-emulsion may be diluted by addition ofa suspension of an active ingredient such as a drug or an immunogen,preferably an immunogen, to obtain the final composition. Alternatively,the pre-emulsion may be diluted with an aqueous solution and used todissolve a dried product such as a dry product.

The immunogen or antigen suitable for use in the present invention maybe selected from the group consisting of inactivated pathogens,attenuated pathogens, immunogenic sub-units (e.g. proteins,polypeptides, peptides, epitopes, haptens), or recombinant expressionvectors, including plasmids having immunogenic inserts. In oneembodiment of the present invention, the immunogen is an inactivated orkilled microorganism. In another embodiment of the invention, thevaccine composition comprises an immunogen selected from the group ofavian pathogens including, but not limited to, Salmonella typhimurium,Salmonella enteritidis, Infectious Bronchitis virus (IBV), NewcastleDisease virus (NDV), egg drop syndrome virus (EDS), or Infectious BursalDisease virus (IBDV), avian influenza virus, and the like, andcombinations thereof.

Alternately, the vaccine composition comprises an immunogen selectedfrom a feline pathogen such as, but not limited to, feline herpesvirus(FHV), feline calicivirus (FCV), feline leukemia virus (FeLV), felineimmunodeficiency virus (FIV), rabies virus, and the like, andcombinations thereof.

In yet another embodiment, a vaccine composition of the presentinvention comprises an immunogen selected from a canine pathogenincluding, but not limited to, rabies virus, canine herpesvirus (CHV),canine parvovirus (CPV), canine coronavirus, Leptospira canicola,Leptospira icterohaemorragiae, Leptospira grippotyphosa, Borreliaburgdorferi, Bordetella bronchiseptica and the like, and combinationsthereof.

In yet another embodiment of the invention the composition comprises animmunogen selected from an equine pathogen, such as equine herpesvirus(type 1 or type 4), equine influenza virus, tetanus, west nile virus,and the like or combinations thereof.

In yet another embodiment of the invention, the composition comprises animmunogen selected from an bovine pathogen, such as foot and mouthdisease virus (FMDV), rabies virus, bovine rotavirus, bovineparainfluenza virus type 3 (bPIV-3), bovine coronavirus, bovine viraldiarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV),Infectious Bovine Rhinotracheitis virus (IBR), Escherichia coli,Pasteurella multocida, Pasteurella haemolytica and the like andcombinations thereof.

In still another embodiment of the present invention, the compositioncomprises an immunogen selected from an porcine pathogen such as, butnot limited to, swine influenza virus (SIV), porcine circovirus type 2(PCV-2), porcine reproductive respiratory syndrome virus (PRRS),pseudorabies virus (PRV), porcine parvovirus (PPV), FMDV, Mycoplasmahyopneumoniae, Erysipelothrix rhusiopathiae, Pasteurella multocida,Bordetella bronchiseptica, Escherichia coli and the like, andcombinations thereof.

Another embodiment of the invention provides for vaccine compositionscomprising at least one immunogen and an emulsion in a pharmaceuticallyacceptable vehicle. Immunogens comprising viruses, bacteria, fungi andthe like may be produced by in vitro culture methods using appropriateculture medium or host cells lines and conventional methods well knownto those of ordinary skill in the art. For example, PRRS may be culturedin an appropriate cell line, such as MA-104 cell line (see U.S. Pat.Nos. 5,587,164; 5,866,401; 5,840,563; 6,251,404 among others). In asimilar manner, PCV-2 may be cultured using PK-15 cells line (see U.S.Pat. No. 6,391,314); SIV may be cultured on eggs (U.S. Pat. No.6,048,537); and Mycoplasma hyopneumoniae may be cultured in aappropriate culture medium (U.S. Pat. No. 5,968,525; U.S. Pat. No.5,338,543; Ross R. F. et al., Am. J. Vet. Res., 1984, 45: 1899-1905).

In order to obtain an inactivated immunologic, or vaccine composition,the pathogen is preferably inactivated after harvesting and, optionally,subjected to clarification by means of a chemical treatment using, forexample, formalin or formaldehyde, beta-propiolactone, ethyleneimine,binary ethyleneimine (BEI), and/or a physical treatment (e.g. a heattreatment or sonication). Methods for inactivation are well known tothose of skill in the art. For example, the FMD virus may be inactivatedby ethyleneimine (Cunliffe, H R, Applied Microbiology, 1973, p. 747-750)or by high pressure (Ishimaru et al., Vaccine 22 (2004) 2334-2339), thePRRS virus may be inactivated by beta-propiolactone treatment(Plana-Duran et al., Vet. Microbiol., 1997, 55: 361-370) or by BEItreatment (U.S. Pat. No. 5,587,164); inactivation of PCV-2 virus may beaccomplished using ethyleneimine treatment or by beta-propiolactonetreatment (U.S. Pat. No. 6,391,314); swine influenza virus may beinactivated using a detergent like Triton, or with formaldehydetreatment (U.S. Pat. No. 6,048,537); Mycoplasma hyopneumoniae bacteriummay be inactivated by formaldehyde treatment (Ross R. F. supra), byethylenimine or BEI treatment (see WO 91/18627).

The inactivated pathogen can be concentrated by conventionalconcentration techniques, in particular by ultrafiltration, and/orpurified by conventional purification means, in particular usingchromatography techniques including, but not limited to, gel-filtration,ultracentrifugation on a sucrose gradient, or selective precipitations,in particular in the presence of polyethylene glycol (PEG).

Immunogens useful in vaccine compositions according to the presentinvention also include expression vectors. Such vectors include, but arenot limited to, in vivo recombinant expression vectors such as apolynucleotide vector or a plasmid (EP-A2-1001025; Chaudhuri P, Res.Vet. Sci. 2001, 70: 255-6), virus vectors such as, but not limited to,adenovirus vectors, poxvirus vectors such as fowlpox (U.S. Pat. Nos.5,174,993; 5,505,941; and 5,766,599) or canarypox vectors (U.S. Pat. No.5,756,103) or bacterial vectors (Escherichia coli or Salmonella sp.)/

The present invention also encompasses the formulation of multivalentimmunological compositions or combination vaccine compositions. Forexample, antigens useful in a combination bovine bacterin made accordingto the present invention include, but are not limited to, Mycoplasmabovis, Pasteurella sp., particularly P. multocida and P. haemolytica,Haemophilus sp., particularly H. somnus, Clostridium sp., Salmonella,Corynebacterium, Streptococcus, Staphylococcus, Moraxella, E. coli andthe like.

The present invention further provides for methods for inducing animmune response in a host, e.g., an animal, comprising administering tothe host an immunological composition or a vaccine composition accordingto the invention. The immune responses elicited in this manner arenotably antibody and/or cellular immune responses, and in particular, agamma-interferon response.

In particular, the present invention provides for methods to immunizeagainst, or to prevent or to reduce the symptoms caused by, infection ofan animal with a pathogenic organism (for example, infection by a virus,bacteria, fungus, or protozoan parasite). The method of the presentinvention is useful in vertebrate animals including, but not limited to,humans, canines (e.g., dogs), felines (e.g., cats); equines (e.g.,horses), bovines (e.g., cattle) and porcine animals (e.g., pigs), aswell as in avians including, but not limited to, chickens, turkeys,ducks, geese, a quail, a pheasant, parrots, finches, hawks, crows andratites (ostrich, emu, cassowary, and the like).

In a particular aspect of the invention, these methods consist of thevaccination of pregnant females before parturition by administering avaccine composition made according to the invention. These methodsfurther include the induction of protective antibodies elicited by thevaccination protocol and the transfer of these protective antibodiesfrom vaccinated pregnant females to their offspring. The transfer ofsuch maternal antibodies subsequently protects the offspring fromdisease.

The dosage of the vaccine composition made according to the presentinvention will depend on the species, breed, age, size, vaccinationhistory, and health status of the animal to be vaccinated. Other factorslike antigen concentration, additional vaccine components, and route ofadministration (i.e., subcutaneous, intradermal, oral, intramuscular orintravenous administration) will also impact the effective dosage. Thedosage of vaccine to administer is easily determinable based on theantigen concentration of the vaccine, the route of administration, andthe age and condition of the animal to be vaccinated. Each batch ofantigen may be individually calibrated. Alternatively, methodicalimmunogenicity trials of different dosages, as well as LD₅₀ studies andother screening procedures can be used to determine effective dosage fora vaccine composition in accordance with the present invention withoutundue experimentation. From the examples presented below, it will bereadily apparent what approximate dosage and what approximate volumewould be appropriate for using the vaccine composition described herein.The critical factor is that the dosage provides at least a partialprotective effect against natural infection, as evidenced by a reductionin the mortality and morbidity associated with natural infection. Theappropriate volume is likewise easily ascertained by one of ordinaryskill in the art. For example, in avian species the volume of a dose maybe from about 0.1 ml to about 0.5 ml and, advantageously, from about 0.3ml to about 0.5 ml. For feline, canine and equine species, the volume ofa dose may be from about 0.2 ml to about 3.0 ml, advantageously fromabout 0.3 ml to about 2.0 ml, and more advantageously, from about 0.5 mlto about 1.0 ml. For bovine and porcine species, the volume of dose maybe from about 0.2 ml to about 5.0 ml, advantageously from about 0.3 mlto about 3.0 ml, and more advantageously from 0.5 ml to about 2.0 ml.

Repeated vaccinations may be preferable at periodic time intervals toenhance the immune response initially or when a long period of time haselapsed since the last dose. In one embodiment of the present invention,the vaccine composition is administered as a parenteral injection (i.e.,subcutaneously, intradermally, or intramuscularly). The composition maybe administered as one dose or, in alternate embodiments, administeredin repeated doses of from about two to about five doses given atintervals of about two to about six weeks, preferably from about two toabout five weeks. However, one of skill in the art will recognize thatthe number of doses and the time interval between vaccinations dependson a number of factors including, but not limited to, the age of theanimal vaccinated; the condition of the animal; the route ofimmunization; amount of antigen available per dose; and the like. Forinitial vaccination, the period will generally be longer than a week andpreferably will be between about two to about five weeks. For previouslyvaccinated animals, a booster vaccination, before or during pregnancy,at about an annual interval may be performed.

The present invention also contemplates administering a vaccinecomposition using a needlefree injector such as PIGJET®, AVIJET®,DERMOJET® or BIOJECTOR® (Bioject, Oregon, USA). An person of ordinaryskill in the art is able to adjust the specifications of the injector asrequired with regard to factors such as the species of the animal to bevaccinated; the age and weight of the animal, and the like without undueexperimentation.

In one embodiment of the present invention, the method comprises asingle administration of a vaccine composition formulated with anemulsion according to the invention. For example, in one embodiment, thevaccine composition is an inactivated FMD virus composition, while analternate embodiment provides for a vaccine comprising an inactivatedPCV2 virus composition. Other immunological compositions or vaccines aresuitable for use in a single dose regimen including, but not limited to,inactivated Mycoplasma hyopneumoniae, PRRS and SIV.

The invention further relates to methods to treat a host, e.g., ananimal, comprising administering to the host a pharmaceuticalcomposition made according to the invention and comprising at least oneimmunogen selected from the group consisting of proteins or peptides,inactivated or attenuated virus, antibodies, allergens, CpG ODN, growthfactors, cytokines, or antibiotics, and in particular CpG ODN orcytokines These pharmaceutical compositions can be used to improvegrowth performances in an animal such as a chicken, a pig, a cow orcattle.

The present invention further relates to a kit comprising a single vialcontaining an ingredient such as a purified immunogen combined with anemulsion made according to the present invention. The kit canalternatively comprise a first vial containing an ingredient such as animmunogen or pharmaceutical composition, combined with saponin andaluminum hydroxide, and a second vial containing an emulsion madeaccording to the present invention. The immunogen may be in a dry form,a dried form or in aqueous solution as described herein.

The invention will now be further described by way of the followingnon-limiting examples.

EXAMPLE 1 Emulsion Manufacturing Method

The emulsion was prepared by Inversion method. In a first step, theaqueous phase and the oily phase were mixed together at +40° C. In asecond step, the emulsion was cooled progressively below the PIT at +5°C. in order to obtain an O/W emulsion. After inversion, the finalemulsion (i.e. vaccine formulation) was mixed and subsequently stored at+5° C. (summarized in Table 1).

TABLE 1 Percent for Percent Each Phase Total (v/v) Incomplete Emulsion -Oily phase (120 mL): 33 Sorbitan monooleate (SPAN 80 ®)  1.8% w/vSorbitan trioleate (20 OE) (TWEEN 85 ®)  10.2% w/v Paraffin oil (MARCOL82 ®)   88% v/v Incomplete Emulsion - Aqueous phase #1 33 (120 mL): 20%(w/v) solution of sorbitan monooleate 11.25% w/v (20 OE) (TWEEN 80 ®)Phosphate disodic and monopotassic 0.02M 85.75% v/v isotonic buffer (pH7.8) Incomplete Emulsion = Oily + Aqueous #1 66 Ratio IncompleteEmulsion/Final Emulsion (i.e. ⅔ Vaccine Formulation) Add Aqueous phase#2 (120 mL): 33 [Phosphate disodic and monopotassic 0.02M isotonicbuffer pH 7.8, saponin, aluminum hydroxide, antigens, M102]* IncompleteEmulsion (Oily + Aq1) + Aq2 = 100 Final Emulsion (i.e. VaccineFormulation) *Concentration/Amount Ranges Aluminum hydroxide - fromabout 0.0% to about 1.0% (w/v), with respect to the vaccine formulationvolume Saponin - from about 0.1 mg to about 2 mg per mL vaccineformulation Antigens - from about 0.1 μg to about 200 μg per mL vaccineformulation M102 & Phosphate to volume

Sorbitan monooleate (SPAN 80®) and sorbitan trioleate (20 OE) (TWEEN85®) were introduced in the oily phase. The sorbitan monooleate (20 OE)(TWEEN 80®) was not miscible in the paraffin oil. A 20% (w/v) solutionof TWEEN 80® was prepared in the same buffer as the vaccine, forexample, in phosphate disodic and monopotassic 0.02M isotonic buffer (pH7.8). When the agitation stopped, the emulsion changed to anoil-in-water emulsion. The emulsion was placed in a cold chamber at 5°C. for at least 4 hours. At this stage, the emulsion was a pre-emulsioncontaining 50% of oily phase.

Second Step: The aqueous phase #2 was prepared with 120 ml of phosphatedisodic and monopotassic 0.02M isotonic buffer pH 7.8 with immunogens(inactivated FMDV, Mycoplasma hyopneumoniae immunogen, or PCV-2immunogen, as described infra), saponin, and aluminum hydroxide. Thepre-emulsion as prepared in the first step was cooled to about 5° C.,diluted by adding half the volume of the aqueous phase #2 at the sametemperature, and mixed by the rotation of a magnetic bar for 1 minute.Final surfactant concentration in the TSAP emulsion was 4.75% (w/v).

In general, the components of the vaccine formulations disclosed hereinwere added in the following order: 1) Media 102 at 5° C., 2) Saponin, 3)Alumine hydroxide, 4) Antigens, and 5) Incomplete emulsion (i.e. thecombination of the Oily Phase plus the Aqueous Phase #1). As preparedherein, the TSAP vaccines are stable for up to 36 months at 5° C.

Using the same preparation method, other emulsions can be obtained asdescribed in the prophetic examples below:

TSAP-2 Emulsion

The TSAP-2 emulsion is an O/W emulsion containing 33% of an oily phase.The oily phase (120 ml) contains MARCOL 82® 88% v/v, SPAN 80® 1.8% w/vand TWEEN 85® 10.2% w/v. The aqueous phase #1 (120 ml) containsphosphate disodic and monopotassic 0.02M isotonic buffer (pH 7.8) 97.75%v/v, and LUTROL F127® 0.75% w/v. The aqueous phase #2 (120 ml) isconstituted with the phosphate disodic and monopotassic 0.02M isotonicbuffer (pH 7.8), saponin, aluminum hydroxide, and optionally containingimmunogens. Final surfactant concentration in the TSAP-2 emulsion isabout 4.25% w/v.

TSAP-3 Emulsion

The TSAP-3 emulsion is an O/W emulsion containing 50% of an oily phase.The oily phase (160 ml) contains MARCOL 82® 92% v/v, SPAN 85® 1.8% w/vand BRIJ 96® 6.2% w/v. The aqueous phase #1 (160 ml) contains phosphatedisodic and monopotassic 0.02M isotonic buffer (pH 7.8) 98.5% v/v, andLUTROL F127® 0.5% w/v, saponin, aluminum hydroxide, and optionallycontaining immunogens. Final surfactant concentration in the TSAP-3emulsion is about 4.25% w/v.

TSAP-4 Emulsion

The TSAP-4 emulsion is an O/W emulsion containing 10% of an oily phase.The oily phase (120 ml) contains MARCOL 82® 60% v/v, SPAN 40® 17.2% w/vand ARLATONE 650® 22.8% w/v. The aqueous phase #1 (120 ml) containsphosphate disodic and monopotassic 0.02M isotonic buffer (pH 7.8) 97.5%v/v and TWEEN 20® 2.5% w/v. The aqueous phase #2 was prepared with 400ml of phosphate disodic and monopotassic 0.02M isotonic buffer pH 7.8,saponin, aluminum hydroxide, and optionally containing immunogens. 100ml of the pre-emulsion was diluted with the 400 ml of the aqueous phase#2 to obtain the TSAP-3 emulsion. Final surfactant concentration in theTSAP-4 emulsion is 4.25% w/v.

EXAMPLE 2 Determination of the Phase Inversion Temperature (PIT) of anEmulsion

10 ml of the TSAP emulsion was placed into a glass tube in a water-bathat a temperature of about 25° C. The TSAP emulsion was a whitehomogeneous emulsion. The temperature in the water bath wasprogressively increased. Changes in the emulsion were visually observed(the emulsion became two separated phases due to the migration of theyellow-brown oily phase to the surface). This change is characteristicof the breakdown of the emulsion. The temperature at which this changeis observed is the PIT value of the emulsion. For the TSAP emulsion, thePIT ranges from about 36° C.-46° C. FIGS. 1-5 provide PIT determinationgraphs for vaccine formulations made according to the present invention(1 year stability study). FIG. 6 provides a PIT determination graph forvaccine formulations made according to the present invention and storedfor 36 months (3 year stability study).

EXAMPLE 3 Study #1: Stability of Vaccine Formulations Prepared Accordingto Example 1

This table indicates the stability (i.e. the time in months theformulations remain as oil in water emulsions) of vaccine formulationsprepared according to the method described in Example 1. Theformulations are comprised of the indicated constituent ingredients (seeformulations 1-13), and the antigens used for each of these formulationscomprised inactivated FMD virus isolates that were considered moderatelyto highly purified.

As Table 2 indicates, the presence of Aluminum hydroxide providesincreased vaccine stability especially when the highest concentration ofantigen is used (compare the enhanced stability of formulations 9, 11,and 13 to the relatively reduced stability at 12 months of formulations3, 5, and 7). On average, the presence of Aluminum hydroxide in thehigher antigen-containing formulations (i.e. formulations 9, 11, and 13)increases the oil/water stability time from about 3-6 months (i.e. theoil/water stability observed for formulations 3, 5, and 7) to abouttwelve (12) months (i.e. the oil/water stability observed forformulations 9, 11), or even to about twenty four (24) months (i.e. theoil/water stability observed for formulation 13).

TABLE 2 Trial 1 2 3 4 5 6 7 8 9 10 11 12 13 Antigen (μg/dose) 0 15 60 1560 15 60 15 60 15 60 15 60 Saponin (mg/dose) 0 0 0 0.5 0.5 1 1 0 0 0.50.5 1 1 Al(OH)₃ (% final) 0 0 0 0 0 0 0 0 .3 .3 .3 .3 .3 Number doses 7575 75 75 75 75 75 75 75 75 75 75 75 T0 Mos O/W O/W O/W O/W O/W O/W O/WO/W O/W O/W O/W O/W O/W T3 Mos O/W O/W O/W O/W O/W O/W O/W O/W O/W O/WO/W O/W O/W T6 Mos O/W O/W W/O O/W W/O O/W O/W O/W O/W O/W O/W O/W O/WT9 Mos O/W O/W W/O O/W W/O O/W W/O O/W O/W O/W O/W O/W O/W T12 Mos O/WO/W W/O O/W W/O O/W W/O O/W O/W O/W O/W O/W O/W T24 Mos O/W O/W W/O O/WW/O OW/ W/O O/W W/O O/W W/O O/W O/W

EXAMPLE 4 Study #2: Stability of Vaccine Formulations Prepared Accordingto Example 1

Vaccine formulations were prepared as described in Example 1 andaccording to the component amounts indicated in Table 3. As describedpreviously, the order of addition of components was 1) media 102, 2)saponin, 3) alumine hydroxide, 4) purified antigens, and 5) incompleteemulsion (refer to Example 1 wherein the incomplete emulsion is definedas the oily phase plus the aqueous phase #1). The purified antigens wereFMDV antigens O1 Campos, A24 Cruzeiro, and C3 Indaial. The total volumewas adjusted using Media 102 (M102, recipe indicated below).

TABLE 3 Trial Number 1 2 3 4 5 6 7 8 9 10 Incomplete emulsion (ml) 400400 400 400 400 400 400 400 400 400 Aqueous phase (ml) 200 200 200 200200 200 200 200 200 200 saponin 0 0 250 9570 0 250 9570 0 250 95704MIL1R159B (mg) O1(ml) [about 1500 μg] 0 18.8 18.8 18.8 18.8 18.8 18.818.8 18.8 18.8 A24 (ml) [about 1500 μg] 0 16.7 16.7 16.7 16.7 16.7 16.716.7 16.7 16.7 C3 (ml) [about 1500 μg] 0 15 15 15 15 15 15 15 15 15Alumine hydroxide 0 0 0 0 25 25 25 150 150 150 1.5% (ml) Media 102 QSP200 ml Total volume (ml) 600 600 600 600 600 600 600 600 600 600 Totalvolume (doses) 300 300 300 300 300 300 300 300 300 300

TABLE 4 For 1 L of Stock Component Description M102 Solution MgCl₂ 95170 mM KCl 190 400 mM CaCl₂ 238 260 mM CHCl₃ 5 ml pure Alanine 276 g —Glucose 1880 4500 mM  Sodium Bicarbonate 2180 2540 mM  Peptone 3000 g —Lactalbumin hydrolysate 4000 g —

Vaccine stability was determined according to Table 5.

TABLE 5 Test Acceptability criteria Droplet size analysis** Mode < 0.18μm; SPAN < 0.8* Phase inversion temperature >=36° C. (PIT) *For thevaccines without alumine gel **Controls performed at the end of thestability study

For each vaccine formulation, the phase inversion temperature (PIT) wasdetermined as described in Example 2. The amount of antigen added isindicated by “hemagluttinin units” (UH) per vaccine dose. Table 6summarizes the PIT data and indicates that the PIT remained generallystable for up to 36 months. FIG. 6 provides graphs of the PITdetermination by conductivity for the vaccine formulations of Trials1-10 (summarized in Table 6). The increase of the PIT that is observedwith the saponin-adjuvanted formulations (i.e. trials 3, 4, 6, 7, 9, and10) constitutes by itself an improvement with respect to stability overnon-saponin-adjuvanted formulations (i.e. trials 2, 5, and 8).

TABLE 6 Trials 1 2 3 4 5 6 7 8 9 10 Days 7 7 7 8 7 9 9   9 14 14 22 2223 23 24 24 29  38 38 41 121 121 121 122 121 121 127  123 133 133 330330 330 340 340 340 340  340 340 340 1100 1100 1100 1100 1100 1100 11001100 1100 1100 Antigen − + + + + + + + + + Saponin 0 0 0.8 2.5 0 0.8 2.5  0 0.8 2.5 (mg/dose) Alumine gel 0 0 0 0 0.065 0.065 0.065   0.39 0.390.39 (%) P.I.T. 39 36 39 39.5 36 39 39.5  37.5 40 40 32.5(*) 36.5 39 4036.5 39 39.5  37.5 39.5 40.5 35 36.5 36.7(*) 39.5 37 39 40  32(*) 4040.5 36.5 37 39 39 37 39 39  38 40 39.5 38.5 39.0 40.5 40 39 40.5 40  4141.5 40.5

The particle size distribution (Table 7) of the emulsions remainedwithin the range set forth in the study criteria over the 36 month timeperiod.

TABLE 7 Alumine Date of Date of Ag Saponin gel Mode Samplesmanufacturing analysis (+/−) (mg/dose) (% final) (μm) SPAN Incompleteemulsion 04/07/05 05/07/05 − 0 0 0.158 0.644 Trial 1 27/09/05 04/12/09 −0 0 0.146 0.650 Trial 2 27/09/05 04/12/09 + 0 0 0.145 0.661 Trial 327/09/05 04/12/09 + 0.8 0 0.148 0.633 Trial 4 27/09/05 04/12/09 + 2.5 00.154 0.582 Trial 5 27/09/05 04/12/09 + 0 0.065 0.144 0.670 Trial 627/09/05 04/12/09 + 0.8 0.065 0.144 0.672 Trial 7 27/09/05 04/12/09 +2.5 0.065 0.144 0.672 Trial 8 27/09/05 05/12/09 + 0 0.39 0.146 0.790Trial 9 27/09/05 05/12/09 + 0.8 0.39 0.145 0.789 Trial 10 27/09/0505/12/09 + 2.5 0.39 0.145 0.787

EXAMPLE 5 Serology Results After Administration of Multiple Doses of aFoot-And-Mouth Disease (FMD) Virus Vaccine Adjuvanted with TSAPEmulsion—Bovine Study

Materials and Methods: 90 Cattle, 12 to 14 months old, never vaccinated,and without FMD antibodies were selected, randomized, and distributedamong 10 groups of 9 animals to be vaccinated. The animals werevaccinated with the indicated vaccine formulations (9807-9814) at day 1.Each of the formulations listed in Tables 8, 9, 10, and 11 was preparedaccording to Example 1 and comprises all the components recited inExample 1 with the saponin and aluminum hydroxide amounts varyingaccording to the following: the amount of saponin is either 0, 0.7, 1.3,or 2.7 mg/dose, and the amount of Algel aluminum hydroxide is either 0%or 0.37%. Eight (8) groups were vaccinated by the intramuscular route(the IM group) and two (2) groups were vaccinated by the subcutaneousroute (the SC group). Each animal of the IM group was revaccinated withthe respective vaccine on day 56 by the intramuscular route and on day84 by the subcutaneous route. Each animal of the SC group wasrevaccinated with the respective vaccine on days 56 and 84 by thesubcutaneous route.

Table 8 summarizes the stability of the vaccine formulations.

TABLE 8 6° C. Formulation 2 Environment Number 1 Week Weeks 30 Days 1Week 2 Weeks 30 Days 9807 OK OK OK OK OK OK 9808 OK OK OK OK OK OK 9809OK OK OK OK OK OK 9810 OK OK OK OK OK OK 9811 OK OK OK OK OK OK 9812 OKOK OK OK OK OK 9813 OK OK OK OK OK OK 9814 OK OK OK OK OK OK

There was not any lesion after the first vaccination by theintramuscular route. There were some lesions after the secondvaccination by the intramuscular route but all these lesions receded 28days after the second vaccination by the intramuscular route. Table 9summarizes the safety of the vaccine formulations, as indicated byanimal body weight measurement on the specified dates.

TABLE 9 Body Weight Measurement Date 17- 8- 15- 12- 15- 9- 12- 16- 9-Experiment Nov- Dec- Dec- Jan- Jan- Feb- Feb- Feb- Mar- IdentifierStatistics 08 08 08 09 09 09 09 09 09 EXP 9807 Mean Wt (kg) 294 303 305331 330 354 352 355 372 StdDev 33 30 31 35 33 34 34 32 37 EXP 9808 MeanWt (kg) 293 304 305 331 328 352 355 354 370 StdDev 34 36 35 35 41 39 4241 42 EXP 9809 Mean Wt (kg) 293 304 304 327 325 351 345 348 365 StdDev33 33 32 29 33 29 37 35 37 EXP 9810 Mean Wt (kg) 293 305 307 330 330 350348 351 374 StdDev 33 32 30 39 36 37 34 34 40 EXP 9811 G1 Mean Wt (kg)295 307 307 328 329 352 346 352 369 StdDev 34 31 31 31 34 36 35 40 39EXP 9811 G2 Mean Wt (kg) 293 304 305 328 329 346 342 345 364 StdDev 3132 34 34 37 41 39 41 43 EXP 9812 G1 Mean Wt (kg) 294 307 307 329 331 352350 354 371 StdDev 29 33 34 35 35 37 36 35 32 EXP 9812 G2 Mean Wt (kg)292 304 304 330 329 352 349 355 369 StdDev 30 32 32 34 37 35 32 37 33EXP 9813 Mean Wt (kg) 293 304 304 332 331 357 354 360 378 StdDev 30 2727 28 30 33 30 30 35 EXP 9814 Mean Wt (kg) 293 304 305 327 325 348 343346 365 StdDev 30 31 29 31 30 32 35 31 29

Results: Table 10 summarizes the serological data collected during theexperimental trials. O1 Campos, A24 Cruzeiro, and C3 Indaial are threeindependent serotypes of the FMD virus, and the presence of O1, A24, andC3 antibodies is a positive indicator that the vaccine formulationelicited an immune response in the vaccinates. “G1” indicates thevaccine formulation was administered intramuscularly and “G2” indicatesthe vaccine formulation was administered subcutaneously. For the presentinvention, particularly for the FMD viral antigens, there is a strong,direct correlation between antibody titer (i.e. serum levels of O1Campos, A24 Cruzeiro, and C3 Indaial) and the Equivalent PopulationProtection (EPP) number. Simply stated, when the antibody titers arehigh, the vaccinate animals are correspondingly highly protected fromviral infection.

Surprisingly, the presence of aluminum hydroxide is associated with asignificant difference in immune response that depends upon the route ofadministration. When aluminum hydroxide is present, as in formulations9812G1 and 9812G2, there is a significant increase in the vaccinateimmune response, as measured by the antibody titers, when the vaccineformulation is administered subcutaneously. There is no similarsignificant difference in efficacy due to route of administration forthe corresponding vaccine formulations that do not contain aluminumhydroxide, namely 9811G1 and 9811G2.

The reason for this enhanced immune response due to the presence ofaluminum hydroxide coincident with the use of the subcutaneous route ofadministration is not known at this time, but an effective embodiment ofthe current invention could include, but is in no way limited to,varying the route of vaccine administration dependent upon current andfuture efficacy (e.g. antibody titer) data.

As compared to non-saponin-adjuvanted control vaccine formulations (i.e.9807 & 9808), the saponin-adjuvanted vaccine formulations elicit in thestudy animals a more rapid immune response to all three FMD virusantigens, as indicated by the higher average antibody titers forsaponin-adjuvanted formulations at Day 21. Taken together, the evidenceindicates that the present invention provides improved stability andenables more rapid immune responses than non-saponin adjuvantedformulations.

TABLE 10 Saponin O1 CAMPOS A24 CRUZEIRO C3 INDAIAL MEANS (mg/ Al(OH)₃D21 D56 D21 D56 D21 D56 D21 D56 dose (%) Form. ID TIT EPP TIT EPP TITEPP TIT EPP TIT EPP TIT EPP TIT TIT 0 0 9807 1.47 37.7 2.07 80.4 1.5629.4 2.01 71.6 1.97 69.5 2.41 92.3 1.67 2.2 0 0.37 9808 1.64 51.3 1.5945.6 1.9 55.1 1.66 30.5 2.08 77.3 2.14 85.8 1.88 1.8 0.7 0 9809 1.6649.4 1.91 64.9 1.97 61.5 1.9 52.8 2.05 77.4 2.37 87.7 1.89 2.1 0.7 0.379810 1.91 71.3 1.82 64.5 2.02 65.7 1.81 46.9 2.27 88.7 2.28 88 2.07 21.3 0 9811 G1 1.97 74.6 2.18 82.6 2.03 67.8 2.01 70.1 2.32 88.6 2.4692.2 2.11 2.2 1.3 0 9811 G2 1.92 73.4 2.06 84 2.05 68.2 2.11 74 2.3392.2 2.55 95.3 2.1 2.2 1.3 0.37 9812 G1 1.81 62.5 1.98 67.3 2.08 73.41.88 49.2 2.29 88.5 2.27 87.4 2.06 2 1.3 0.37 9812 G2 2.58 98.9 2.42 972.5 94.5 2.29 90 2.59 96.7 2.76 98.8 2.56 2.5 2.7 0 9813 2 79.5 2.3193.5 2.22 87.5 2.23 88.6 2.43 93.4 2.69 96.5 2.22 2.4 2.7 0.37 9814 2.1488.2 2.03 80.2 2.34 95.1 1.96 57.4 2.6 96.4 2.39 92.5 2.36 2.1 G1 -vaccine formulation was administered intramuscularly G2 - vaccineformulation was administered subcutaneously

EXAMPLE 6 Serology Results after Administration of Two Doses of aFoot-And-Mouth Disease (FMD) Virus Vaccine Adjuvanted with TSAPEmulsion—Porcine Study

Materials and Methods: 57 Pigs, never vaccinated, and without FMDantibodies were selected, randomized, and distributed among 9 groups of6 animals to be vaccinated and 1 group of 3 animals not to be vaccinated(non-vaccinated animals). The animals were vaccinated with either theindicated vaccine formulations (A-I) on day 1. Each of the formulationslisted in Table 11 was prepared according to Example 1 and comprises allthe components recited in Example 1 with the saponin and aluminumhydroxide varying according to the following: the amount of saponin iseither 0, 0.7, 1.3, or 2.7 mg/dose and the amount of Algel aluminumhydroxide is either 0% or 0.37%. Vaccine formulation safety was assessedvia measurement of several parameters including rectal temperature andvisual indicia of the animal's reaction to vaccination.

TABLE 11 Vaccine Formulation A B C D E F G H I Ag (μg/dose) 0 10 10 1010 10 10 10 10 Saponin 0 0 0 0.7 0.7 1.3 1.3 2.7 2.7 (mg/dose) Algel (%final) 0 0 0.37 0 0.37 0 0.37 0 0.37 Incomplete 333 333 333 333 333 333333 333 333 Emulsion Aqueous Phase 2 (166 mL) Purified FMDV 0 11.1 11.111.1 11.1 11.1 11.1 11.1 11.1 (mL) Saponin (mL) 0 0 0 2.48 2.48 4.964.96 9.92 9.92 Algel 3% (mL) 0 0 61.7 0 61.7 0 61.7 0 67.7 M102 at 5° C.166 154.9 93.2 152.4 90.8 149.9 88.2 145 83.3 Total Volume total 500 500500 500 500 500 500 500 500 (mL) Number total 250 250 250 250 250 250250 250 250 (doses)

Results: All vaccine formulations that contained antigen (formulationsB-I, described in Table 11) induced in the test animals an antibodyresponse that was significantly greater than that induced by the noantigen control (formulation I, described in Table 11). Formulationsthat contained saponin induced relatively greater antibody responses ascompared to formulations that contained no saponin. Table 12 summarizesthe antibody titer data, and restates the amounts of antigen, saponin,and aluminum hydroxide for each formulation. In particular, the antibodytiter data indicates that the presence in vaccine formulations ofbetween about 0.7 mg/dose and about 2.7 mg/dose saponin increases theantibody response, relative to vaccine formulations that do not containsaponin.

TABLE 12 Antigen Saponin Average Animal Vaccine (μg/ (mg/ Antibody TiterData Group Formulation dose) dose) Algel Statistic D21 D44 G1 A  0 0 0Mean 0.75 0.67 StdDev 0.26 0.26 G2 B 10 0 0 Mean 1.45 3.17 StdDev 0.500.48 G3 C 10 0 0.37 Mean 1.40 2.92 StdDev 0.18 0.31 G4 D 10 0.7 0 Mean1.60 3.12 StdDev 0.31 0.42 G5 E 10 0.7 0.37 Mean 1.87 3.20 StdDev 0.690.45 G6 F 10 1.3 0 Mean 1.40 3.41 StdDev 0.37 0.27 G7 G 10 1.3 0.37 Mean1.80 3.37 StdDev 0.22 0.43 G8 H 10 2.7 0 Mean 1.72 3.47 StdDev 0.66 0.30G9 I 10 2.7 0.37 Mean 1.47 2.97 StdDev 0.18 0.57 G10 Not — — — Mean 0.670.67 Vaccinated StdDev 0.17 0.09

EXAMPLE 7 Determination of the Protective Dose 50% (PD50) of ThreeExperimental FMD Vaccines in Pigs

Overview. The purpose of the study was to test the efficacy against FMDvirulent challenge in pigs of 3 experimental FMD vaccine formulationsaccording to the instant invention. Four vaccines (A, B, C, and D) wereprepared with respectively no Antigen (A), and variable doses ofinactivated purified FMD O1 Manisa antigen (vaccines B, C, and D)formulated with adjuvant according to the instant invention. Thevaccines were presented as 30 mL vials filled to 25 mL and the doseswere 2 mL per dose. The challenge strain was FMD O1 Manisa, 4494024D:911, 20-07-1994 (which had been originally isolated as O1 ManisaTurkey 1/78), and the titer in secondary porcine kidney cells was 7.67log 10 TCID50/mL. Diluent for the challenge strain was Hanks MEM, 2%fetal bovine serum (FBS) with standard antibiotics.

Procedure. Porcines free from FMDV and not previously vaccinated againstFMD were used for the study. Each of 47 pigs aged 10 weeks (plus orminus 1 week) was acclimatized for at least 24 hours prior toexperimental procedures. Pigs were housed 2 to 3 animals per room, andwere fed standard pellet feed and watered ad libitum. At Day 1 (D1), thepigs (regardless of sex) were allocated to 15 groups of three animalsand one group of two animals. Each group was housed in one particularroom and in each room, the animals were individually separated by woodenplanks (1.5M) from challenge until completion of the study.

On D0, animals were vaccinated, intramuscularly behind the ear on theleft side, with individual syringes according to Tables 13 and 14.Before vaccination, vaccine vials were gently inverted about 10 times toensure homogeneous suspension.

TABLE 13 vaccine composition Vaccine (Final Emulsion) A B C D Ag(μg/dose) 0 0.4 2 10 Saponin (mg/dose) 0 0 0 0.7 Algel (% final) 0 0.420.42 0.42 Incomplete Emulsion 333 333 333 333 (ml) Aqueous phase 2 166ml FMD O-Manisa batch# 0 0.66 3.29 16.46 OMAd-04-613 (ml) Pure Saponin 02.48 2.48 2.48 08-0314-P (ml) Alumine Gel 3% (ml) 0 61.4 61.4 61.4 Media102 166 102.5 99.8 86.7 Total Volume total (ml) 500 500 500 500 # total(doses) 250 250 250 250

TABLE 14 vaccination summary Volume of Vaccine injected Group VaccineDose (mL) # Pigs B1 B Dose 2.0 3 B2 B ½ dose 1 3 B3 B ¼ dose 0.5 3 B4 B⅛ dose 0.25 3 B5 B 1/16 dose 0.13 3 C1 C Dose 2.0 3 C2 C ½ dose 1 3 C3 C¼ dose 0.5 3 C4 C ⅛ dose 0.25 3 C5 C 1/16 dose 0.13 3 D1 D Dose 2.0 3 D2D ½ dose 1 3 D3 D ¼ dose 0.5 3 D4 D ⅛ dose 0.25 3 D5 D 1/16 dose 0.13 3A (control) A 1 2 2

Challenge. On D28, FMD type O virus was diluted to obtain 100,000 TCID50per mL (the virus stock was diluted 2.67 log 10, or 468 times). On thesame day, all animals were anesthetized by administration of STRESSNIL(1 mL/20 kg) and KETAMINE (2 mL/20 kg) by the IM route and challengedwith 10,000 TCID50 of virus under 0.1 mL by intra-dermal route, into thebulb of the heel of the outer claw of the left hind foot. The generalwell being of the animals was checked daily from DO to D28. Any clinicalobservations and treatments were noted. After challenge, animals wereobserved daily for 7 day (D29 to D35). Observations were performed inthe same order, starting by the rooms with the animals vaccinated withthe highest to dose to those with the animals vaccinated with the lowestdose, and ending with the controls (group A). The general well being waschecked daily, with particular attention paid to signs of FMD on thesnoot and the feet. Blood was collected from each animal beforevaccination (on D1 or D0), D14, and D28 before challenge and at the endof the study. Samples were heat-inactivated (56° C. for 30 minutes) andFMDV Ab type O titer was determined in all sera by VN test (MERIAL R&D,Lelystad).

On D36 (end of the observation period), animals were euthanized (4 to 6mL per 50 kg IV T61) and closely inspected for signs of FMDV. Anylesions observed in the groups vaccinated with a full dose of vaccine(groups B1, C1, or D1) were samples and frozen at −70° C. for furthervirus typing. Presence of lesions on the snoot, mouth and/or feet(except on the major claw of the inoculated feet) were considered as anevidence of FMD. The test was considered valid as it met the criteria,which stated both control pigs must show FMD clinical signs. The PD50 ofeach vaccine was calculated by Spearman Karber method.

Results. The potency versus payload is presented in FIG. 9, and therelevant study data is numerically summarized in Tables 15 and 16 below.

TABLE 15 # protected pigs per Volume vaccine tested (ml) B C D 2.000 3/32/2 3/3 1.000 2/3 3/3 3/3 0.500 1/3 1/3 3/3 0.250 1/3 1/3 2/3 0.125 0/30/3 1/3

TABLE 16 Calculation of vaccine potency using logistic regressionVaccine Payload (μg) PD50 IC95− IC95+ B 0.4 3.594 1.6046 2.896 C 24.5821 1.7021 2.6979 D 10 11.7 9.533 51.55

1. A vaccine composition comprising an injectable oil-in-water (O/W)emulsion, comprising: (i) an aqueous solution comprising at least oneimmunogen; (ii) an aqueous solution comprising a hydrophilic ionicsurfactant (iii) an optional aqueous solution comprising aluminumhydroxide (iv) a mineral oil; (v) a non-ionic lipophilic surfactant;(vi) a non-ionic hydrophilic surfactant having a highhydrophilic-lipophilic balance (HLB) value between 13 and 40; and (vii)a non-ionic hydrophilic surfactant having a low hydrophilic-lipophilicbalance (HLB) value between 9 and
 13. 2. The composition of claim 1,wherein the hydrophilic ionic surfactant is saponin and wherein theconcentration of the saponin is between about 0.35 mg/dose and about 3.0mg/dose and wherein the concentration of the aluminum hydroxide isbetween about 0% and about 1.0% (w/v).
 3. The composition of claim 2,wherein the high HLB non-ionic hydrophilic surfactant is present at aconcentration of 0.1 to 1.5% expressed as a weight by volume of emulsion(w/v), and wherein the percentage of the surfactants is from about 4% toabout 8% weight/volume.
 4. The composition of claim 2, wherein the lowHLB non-ionic hydrophilic surfactant is present at a concentration of 1%to 8% expressed as a weight by volume of emulsion (w/v).
 5. Thecomposition of claim 2, wherein the non-ionic lipophilic surfactant ispresent at a concentration of 0.1% to 2.5% expressed as a weight byvolume of emulsion (w/v).
 6. The composition of claim 2, wherein themineral oil is present at a concentration of 20% to 40% (v/v), andwherein the emulsion has a phase inversion temperature (PIT) of betweenabout 33° C. to about 66° C.
 7. The composition of claim 2, wherein thelow HLB non-ionic hydrophilic surfactant is selected from the groupconsisting of ethoxylated fatty acid triesters of sorbitan, ethoxylatedfatty acid diesters of sorbitan, ethoxylated fatty acid monoesters ofsorbitan, ethoxylated fatty alcohols, ethoxylated fatty acidsethoxylated castor oil and combinations thereof, and wherein the esterof said ethoxylated fatty acid ester is selected from the groupconsisting of oleate, palmitate, stearate, isostearate, laurate andcombinations thereof.
 8. The composition of claim 2, wherein thenon-ionic lipophilic surfactant is selected from the group consisting offatty acid esters of sorbitan, fatty acid esters of mannide,di-ethoxylated fatty acid esters of mannide, tri-ethoxylated fatty acidesters of mannide, tetra-ethoxylated fatty acid esters of mannide andcombinations thereof, and wherein the ester of the fatty acid esters isselected from the group consisting of oleate, palmitate, stearate,isostearate, laurate and combinations thereof.
 9. The composition ofclaim 2, wherein the high HLB non-ionic hydrophilic surfactant isselected from the group consisting of ethoxylated fatty acid monoestersof sorbitan, ethoxylated fatty alcohols, ethoxylated fatty acids,non-ionic block-copolymer, and combinations thereof, and wherein theethoxylated monoester of sorbitan is selected from the group consistingof ethoxylated sorbitan monolaurate, ethoxylated sorbitan monopalmitate,ethoxylated sorbitan monostearate, ethoxylated sorbitan monooleate, andcombinations thereof.
 10. The composition of claim 2, wherein themineral oil is selected from the group consisting of paraffin oil,squalane, pristane, polyisobutene oil, hydrogenated polyisobutene oil,polydecene oil, polyisoprene oil, polyisopropene oil and, combinationsthereof.
 11. The composition of claim 2 which comprises a paraffin oil,a sorbitan fatty acid monoester as a non-ionic lipophilic surfactant, anethoxylated fatty acid triester of sorbitan as a low HLB non-ionichydrophilic surfactant and a non-ionic block-copolymer as a high HLBnon-ionic hydrophilic surfactant.
 12. The composition of claim 9,wherein the sorbitan fatty acid monoester is a sorbitan monooleate, theethoxylated fatty acid triester of sorbitan is an ethoxylated trioleateof sorbitan and the non-ionic block-copolymer is apolyoxyethylene/polyoxypropylene polymer (POE-POP).
 13. The compositionof claim 12, wherein the paraffin oil is present at a concentration of10% to 40% v/v, the sorbitan monooleate is present at a concentration of0.2% to 1.5% w/v, the ethoxylated trioleate of sorbitan is present at aconcentration of 2% to 5% w/v and the POE-POP is present at aconcentration of 0.1% to 0.5% w/v.
 14. The composition of claim 12,wherein the paraffin oil is present at a concentration of 29.3% v/v, thesorbitan monooleate is present at a concentration of 0.6% w/v, theethoxylated trioleate of sorbitan is present at a concentration of 3.4%w/v and the POE-POP is present at a concentration of 0.25% w/v.
 15. Thecomposition of claim 2, wherein the immunogen is selected from the groupconsisting of an inactivated pathogen, an attenuated pathogen, asubunit, a recombinant expression vector, and a plasmid or combinationsthereof, and wherein the inactivated pathogen is selected from the groupconsisting of a virus, a bacterium, a fungus, protozoal parasite orcombinations thereof.
 16. The composition of claim 15, wherein theimmunogen is an inactivated Foot-and-Mouth disease (FMD) virus, aninactivated porcine circovirus type 2 (PCV-2) virus, or an inactivatedMycoplasma hyopneumoniae bacterium.
 17. A method for inducing animmunological response in an animal against a pathogen comprisingadministering to said animal a vaccine composition according to claim15.
 18. A method according to claim 17, wherein the immunogen isselected from the group consisting of an inactivated pathogen, anattenuated pathogen, a subunit, a recombinant expression vector, and aplasmid or combinations thereof.
 19. A method according to claim 17,wherein the immunogen is selected from the group consisting of aninactivated Mycoplasma hyopneumoniae bacterium, an inactivated porcinecircovirus type 2 (PCV-2) virus or combinations thereof.
 20. The methodof claim 19, wherein the administration is intramuscular (IM),intradermal (ID) or subcutaneous (SC) injection, or wherein theadministration is done with a needleless injector.